Antibody immunization therapy for treatment of atherosclerosis

ABSTRACT

The present invention relates to passive immunization for treating or preventing atherosclerosis using an isolated human antibody directed towards at least one oxidized fragment of apolipoprotein B in the manufacture of a pharmaceutical composition for therapeutical or prophylactical treatment of atherosclerosis by means of passive immunization, as well as method for preparing such antibodies, and a method for treating a mammal, preferably a human using such an antibody to provide for passive immunization.

PRIORITY INFORMATION

This application claims priority to U.S. Provisional Patent Appln. No.60/421,067, filed Oct. 25, 2002, Swedish Patent Application No.0302312-4, filed Sep. 27, 2003 and Swedish Patent Application No.0202959-3, filed Oct. 4, 2002.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to new isolated human antibodies raisedagainst peptides being derivatives of apolipoprotein B, in particularantibodies to be used for immunization therapy for treatment ofatherosclerosis, method for their preparation, and method for passiveimmunization using said antibodies.

In particular the invention includes:

The use of any isolated antibody raised against an oxidized form of thepeptides listed in table 1, in particular MDA-modified peptides,preferably together with a suitable carrier and adjuvant as animmunotherapy or “anti-atherosclerosis “vaccine” for prevention andtreatment of ischemic cardiovascular disease.

2. Description of Prior Art

The protective effects of humoral immunity are known to be mediated by afamily of structurally related glycoproteins called antibodies.Antibodies initiate their biological activity by binding to antigens.Antibody binding to antigens is generally specific for one antigen andthe binding is usually of high affinity. Antibodies are produced byB-lymphocytes. Blood contains many different antibodies, each derivedfrom a clone of B-cells and each having a distinct structure andspecificity for antigen. Antibodies are present on the surface ofB-lymphocytes, in the plasma, in interstitial fluid of the tissues andin secretory fluids such as saliva and mucous on mucosal surfaces.

All antibodies are similar in their overall structure, accounting forcertain similarities in physico-chemical features such as charge andsolubility. All antibodies have a common core structure of two identicallight chains, each about 24 kilo Daltons, and two identical heavy chainsof about 55-70 kilo Daltons each. One light chain is attached to eachheavy chain, and the two heavy chains are attached to each other. Boththe light and heavy chains contain a series of repeating homologousunits, each of about 110 amino acid residues in length which foldindependently in a common globular motif, called an immunoglobulin (Ig)domain. The region of an antibody formed by the association of the twoheavy chains is hydrophobic. Antibodies, and especially monoclonalantibodies, are known to cleave at the site where the light chainattaches to the heavy chain when they are subjected to adverse physicalor chemical conditions. Because antibodies contain numerous cysteineresidues, they have many cysteine-cysteine disulfide bonds. All Igdomains contain two layers of beta-pleated sheets with three or fourstrands of anti-parallel polypeptide chains.

Despite their overall similarity, antibody molecules can be divided intoa small number of distinct classes and subclasses based onphysicochemical characteristics such as size, charge and solubility, andon their behavior in binding to antigens. In humans, the classes ofantibody molecules are: IgA, IgD, IgE, IgG and IgM. Members of eachclass are said to be of the same isotype. IgA and IgG isotypes arefurther subdivided into subtypes called IgA1, IgA2 and IgG1, IgG2, IgG3and IgG4. The heavy chains of all antibodies in an isotype shareextensive regions of amino acid sequence identity, but differ fromantibodies belonging to other isotypes or subtypes. Heavy chains aredesignated by the letters of the Greek alphabet corresponding to theoverall isotype of the antibody, e.g., IgA contains alpha., IgD containsdelta., IgE contains epsilon., IgG contains .gamma., and IgM contains.mu. heavy chains. IgG, IgE and IgD circulate as monomers, whereassecreted forms of IgA and IgM are dimers or pentamers, respectively,stabilized by the J chain. Some IgA molecules exist as monomers ortrimers.

There are between 10⁸ and 10¹⁰ structurally different antibody moleculesin every individual, each with a unique amino acid sequence in theirantigen combining sites. Sequence diversity in antibodies ispredominantly found in three short stretches within the amino terminaldomains of the heavy and light chains called variable (V) regions, todistinguish them from the more conserved constant (C) regions.

Atherosclerosis is a chronic disease that causes a thickening of theinnermost layer (the intima) of large and medium-sized arteries. Itdecreases blood flow and may cause ischemia and tissue destruction inorgans supplied by the affected vessel. Atherosclerosis is the majorcause of cardiovascular disease including myocardial infarction, strokeand peripheral artery disease. It is the major cause of death in thewestern world and is predicted to become the leading cause of death inthe entire world within two decades.

The disease is initiated by accumulation of lipoproteins, primarilylow-density lipoprotein (LDL), in the extracellular matrix of thevessel. These LDL particles aggregate and undergo oxidativemodification. Oxidized LDL is toxic and cause vascular injury.Atherosclerosis represents in many respects a response to this injuryincluding inflammation and fibrosis.

In 1989 Palinski and coworkers identified circulating autoantibodiesagainst oxidized LDL in humans. This observation suggested thatatherosclerosis may be an autoimmune disease caused by immune reactionsagainst oxidized lipoproteins. At this time several laboratories begansearching for associations between antibody titers against oxidized LDLand cardiovascular disease. However, the picture that emerged from thesestudies was far from clear. Antibodies existed against a large number ofdifferent epitopes in oxidized LDL, but the structure of these epitopeswas unknown. The term “oxidized LDL antibodies” thus referred to anunknown mixture of different antibodies rather than to one specificantibody. T cell-independent IgM antibodies were more frequent thanT-cell dependent IgG antibodies.

Antibodies against oxidized LDL were present in both patients withcardiovascular disease and in healthy controls. Although some earlystudies reported associations between oxidized LDL antibody titers andcardiovascular disease, others were unable to find such associations. Amajor weakness of these studies was that the ELISA tests used todetermine antibody titers used oxidized LDL particles as ligand. LDLcomposition is different in different individuals, the degree ofoxidative modification is difficult both to control and assess andlevels of antibodies against the different epitopes in the oxidized LDLparticles can not be determined. To some extent, due to the technicalproblems it has been difficult to evaluate the role of antibodyresponses against oxidized LDL using the techniques available so far,but, however, it is not possible to create well defined and reproduciblecomponents of a vaccine if one should use intact oxidized LDL particles.

Another way to investigate the possibility that autoimmune reactionsagainst oxidized LDL in the vascular wall play a key role in thedevelopment of atherosclerosis is to immunize animals against its ownoxidized LDL. The idea behind this approach is that if autoimmunereactions against oxidized LDL are reinforced using classicalimmunization techniques this would result in increased vascularinflammation and progressive of atherosclerosis. To test this hypothesisrabbits were immunized with homologous oxidized LDL and then inducedatherosclerosis by feeding the animals a high-cholesterol diet for 3months.

However, in contrast to the original hypothesis immunization withoxidized LDL had a protective effect reducing atherosclerosis with about50%. Similar results were also obtained in a subsequent study in whichthe high-cholesterol diet was combined with vascular balloon-injury toproduce a more aggressive plaque development. In parallel with ourstudies several other laboratories reported similar observations. Takentogether the available data clearly demonstrates that there exist immunereactions that protect against the development of atherosclerosis andthat these involves autoimmunity against oxidized LDL.

These observations also suggest the possibility of developing an immunetherapy or “vaccine” for treatment of atherosclerosis-basedcardiovascular disease in man. One approach to do this would be toimmunize an individual with his own LDL after it has been oxidized byexposure to for example copper. However, this approach is complicated bythe fact that it is not known which structure in oxidized LDL that isresponsible for inducing the protective immunity and if oxidized LDLalso may contain epitopes that may give rise to adverse immunereactions.

The identification of epitopes in oxidized LDL is important for severalaspects:

First, one or several of these epitopes are likely to be responsible foractivating the anti-atherogenic immune response observed in animalsimmunized with oxidized LDL. Peptides containing these epitopes maytherefore represent a possibility for development of an immune therapyor “atherosclerosis vaccine” in man. Further, they can be used fortherapeutic treatment of atheroschlerosis developed in man.

Secondly, peptides containing the identified epitopes can be used todevelop ELISAs able to detect antibodies against specific structure inoxidized LDL. Such ELISAs would be more precise and reliable than onespresently available using oxidized LDL particles as antigen. It wouldalso allow the analyses of immune responses against different epitopesin oxidized LDL associated with cardiovascular disease.

U.S. Pat. No. 5,972,890 relates to a use of peptides for diagnosingatherosclerosis. The technique presented in said US patent is as aprinciple a form of radiophysical diagnosis. A peptide sequence isradioactively labelled and is injected into the bloodstream. If thispeptide sequence should be identical with sequences present inapolipoprotein B it will bind to the tissue where there are receptorspresent for apolipoprotein B. In vessels this is above allatherosclerotic plaque. The concentration of radioactivity in the wallof the vessel can then be determined e.g., by means of a gamma camera.The technique is thus a radiophysical diagnostic method based on thatradioactively labelled peptide sequences will bound to their normaltissue receptors present in atherosclerotic plaque and are detectedusing an external radioactivity analysis. It is a direct analysis methodto identify atherosclerotic plaque. It requires that the patient begiven radioactive compounds.

Published studies (Palinski et al., 1995, and George et al., 1998) haveshown that immunisation against oxidised LDL reduces the development ofatherosclerosis. This would indicate that immuno reactions againstoxidised LDL in general have a protecting effect. The results givenherein have, however, surprisingly shown that this is not always thecase. E.g., immunisation using a mixture of peptides #10, 45, 154, 199,and 240 gave rise to an increase of the development of atherosclerosis.Immunisation using other peptide sequences, e.g., peptide sequences #1,and 30 to 34 lacks total effect on the development of atherosclerosis.The results are surprising because they provide basis for the fact thatimmuno reactions against oxidised LDL, can protect against thedevelopment, contribute to the development of atherosclerosis, and bewithout any effect at all depending on which structures in oxidised LDLthey are directed to. These findings make it possible to developimmunisation methods, which isolate the activation of protecting immunoreactions. Further, they show that immunisation using intact oxidisedLDL could have a detrimental effect if the particles used contain a highlevel of structures that give rise to atherogenic immuno reactions.

SUMMARY OF THE INVENTION

The technique of the present invention is based on quite differentprinciples and methods. In accordance with claim 1 the invention relatesto antibodies raised against oxidized fragments of apolipoprotein B,which antibodies are used for immunisation against cardiovasculardisease.

As an alternative to active immunisation, using the identified peptidesdescribed above, passive immunisation with pre-made antibodies directedto the same peptides is an attractive possibility. Such antibodies maybe given desired properties concerning e.g. specificity andcrossreactivity, isotype, affinity and plasma halflife. The possibilityto develop antibodies with predetermined properties became apparentalready with the advent of the monoclonal antibody technology (Milsteinand Köhler, 1975 Nature, 256:495-7). This technology used murinehybridoma cells producing large amounts of identical, but murine,antibodies. In fact, a large number of preclinical, and also clinicaltrials were started using murine monoclonal antibodies for treatment ofe.g. cancers. However, due to the fact that the antibodies were ofnon-human origin the immune system of the patients recognised them asforeign and developed antibodies to them. As a consequence the efficacyand plasma half-lives of the murine antibodies were decreased, and oftenside effects from allergic reactions, caused by the foreign antibody,prevented successful treatment.

To solve these problems several approaches to reduce the murinecomponent of the specific and potentially therapeutic antibody weretaken. The first approach comprised technology to make so calledchimearic antibodies where the murine variable domains of the antibodywere transferred to human constant regions resulting in an antibody thatwas mainly human (Neuberger et al. 1985, Nature 314:268-70). A furtherrefinement of this approach was to develop humanized antibodies wherethe regions of the murine antibody that contacted the antigen, the socalled Complementarity Determining Regions (CDRs) were transferred to ahuman antibody framework. Such antibodies are almost completely humanand seldom cause any harmful antibody responses when administered topatients. Several chimearic or humanized antibodies have been registeredas therapeutic drugs and are now widely used within various indications(Borrebaeck and Carlsson, 2001, Curr. Opin. Pharmacol. 1:404-408).

Today also completely human antibodies may be produced using recombinanttechnologies. Typically large libraries comprising billions of differentantibodies are used. In contrast to the previous technologies employingchimearisation or humanization of e.g. murine antibodies this technologydoes not rely on immunisation of animals to generate the specificantibody. In stead the recombinant libraries comprise a huge number ofpre-made antibody variants why it is likely that the library will haveat least one antibody specific for any antigen. Thus, using suchlibraries the problem becomes the one to find the specific binderalready existing in the library, and not to generate it throughimmunizations. In order to find the good binder in a library in anefficient manner, various systems where phenotype i.e. the antibody orantibody fragment is linked to its genotype i.e. the encoding gene havebeen devised. The most commonly used such system is the so called phagedisplay system where antibody fragments are expressed, displayed, asfusions with phage coat proteins on the surface of filamentous phageparticles, while simultaneously carrying the genetic informationencoding the displayed molecule (McCafferty et al., 1990, Nature348:552-554). Phage displaying antibody fragments specific for aparticular antigen may be selected through binding to the antigen inquestion. Isolated phage may then be amplified and the gene encoding theselected antibody variable domains may optionally be transferred toother antibody formats as e.g. full length immunoglobulin and expressedin high amounts using appropriate vectors and host cells well known inthe art.

The format of displayed antibody specificities on phage particles maydiffer. The most commonly used formats are Fab (Griffiths et al., 1994.EMBO J. 13:3245-3260) and single chain (scFv) (Hoogenboom et al., 1992,J Mol. Biol. 227:381-388) both comprising the variable antigen bindingdomains of antibodies. The single chain format is composed of a variableheavy domain (VH) linked to a variable light domain (VL) via a flexiblelinker (U.S. Pat. No. 4,946,778). Before use as analytical reagents, ortherapeutic agents, the displayed antibody specificity is transferred toa soluble format e.g. Fab or scFv and analysed as such. In later stepsthe antibody fragment identified to have desirable characteristics maybe transferred into yet other formats such as full length antibodies.

Recently a novel technology for generation of variability in antibodylibraries was presented (WO98/32845, Soderlind et al., 2000, NatureBioTechnol. 18:852-856). Antibody fragments derived from this libraryall have the same framework regions and only differ in their CDRs. Sincethe framework regions are of germline sequence the immunogenicity ofantibodies derived from the library, or similar libraries produced usingthe same technology, are expected to be particularly low (Soderlind etal., 2000, Nature BioTechnol. 18:852-856). This property is expected tobe of great value for therapeutic antibodies reducing the risk for thepatient to form antibodies to the administered antibody thereby reducingrisks for allergic reactions, the occurrence of blocking antibodies, andallowing a long plasma half-life of the antibody. Several antibodiesderived from recombinant libraries have now reached into the clinic andare expected to provide therapeutic drugs in the near future.

Thus, when met with the challenge to develop therapeutic antibodies tobe used in humans the art teaches away from the earlier hybridomatechnology and towards use of modern recombinant library technology(Soderlind et al., 2001, Comb. Chem. & High Throughput Screen.4:409-416). It was realised that the peptides identified(PCT/SE02/00679), and being a integral part of this invention, could beused as antigens for generation of fully human antibodies withpredetermined properties. In contrast to earlier art (U.S. Pat. No.6,225,070) the antigenic structures i.e. the peptides used in thepresent invention were identified as being particularly relevant astarget sequences for therapeutic antibodies (PCT/SE02/00679). Also, inthe present invention the antibodies are derived from antibody librariesomitting the need for immunisation of lipoprotein deficient mice toraise murine antibodies (U.S. Pat. No. 6,225,070). Moreover, theresulting antibodies are fully human and are not expected to generateany undesired immunological reaction when administered into patients.

The peptides used, and previously identified (PCT/SE02/00679) are thefollowing:

TABLE 1 A. High IgG, MDA-difference P 11. FLDTVYGNCSTHFTVKTRKG (SEQ IDNO: 39) P 25. PQCSTHILQWLKRVHANPLL (SEQ ID NO: 40) P 74.VISIPRLQAEARSEILAHWS (SEQ ID NO: 41) B. High IgM, no MDA-difference P40. KLVKEALKESQLPTVMDFRK (SEQ ID NO: 42) P 68. LKFVTQAEGAKQTEATMTFK (SEQID NO: 43) P 94. DGSLRHKFLDSNIKFSHVEK (SEQ ID NO: 44) P 99.KGTYGLSCQRDPNTGRLNGE (SEQ ID NO: 45) P 100. RLNGESNLRFNSSYLQGTNQ (SEQ IDNO: 46) P 102. SLTSTSDLQSGIIKNTASLK (SEQ ID NO: 47) P 103.TASLKYENYELTLKSDTNGK (SEQ ID NO: 48) P 105. DMTFSKQNALLRSEYQADYE (SEQ IDNO: 49) P 177. MKVKIIRTIDQMQNSELQWP (SEQ ID NO: 50) C. High IgG, no MDAdifference P 143. IALDDAKINFNEKLSQLQTY (SEQ ID NO: 51) P 210.KTTKQSFDLSVKAQYKKNKH (SEQ ID NO: 52) D. NHS/AHP, IgG-ak > 2,MDA-difference P1. EEEMLENVSLVCPKDATRFK (SEQ ID NO: 53) P 129.GSTSHHLVSRKSISAALEHK (SEQ ID NO: 54) P 148. IENIDFNKSGSSTASWIQNV (SEQ IDNO: 55) P 162. IREVTQRLNGEIQALELPQK (SEQ ID NO: 56) P 252.EVDVLTKYSQPEDSLIPFFE (SEQ ID NO: 57) E. NHS/AHP, IgM-ak > 2,MDA-difference P 301. HTFLIYITELLKKLQSTTVM (SEQ ID NO: 58) P 30.LLDIANYLMEQIQDDCTGDE (SEQ ID NO: 59) P 31. CTGDEDYTYKIKRVIGNMGQ (SEQ IDNO: 60) P 32. GNMGQTMEQLTPELKSSILK (SEQ ID NO: 61) P 33.SSILKCVQSTKPSLMIQKAA (SEQ ID NO: 62) P 34. IQKAAIQALRKMEPKDKDQE (SEQ IDNO: 63) P 100. RLNGESNLRFNSSYLQGTNQ (SEQ ID NO: 64) P 107.SLNSHGLELNADILGTDKIN (SEQ ID NO: 65) P 149. WIQNVDTKYQIRIQIQEKLQ (SEQ IDNO: 66) P 169. TYISDWWTLAAKNLTDFAEQ (SEQ ID NO: 67) P 236.EATLQRIYSLWEHSTKNHLQ (SEQ ID NO: 68) F. NHS/AHP, IgG-ak <0.5, noMDA-difference P 10. ALLVPPETEEAKQVLFLDTV (SEQ ID NO: 69) P 45.IEIGLEGKGFEPTLEALFGK (SEQ ID NO: 70) P 111. SGASMKLTTNGRFREHNAKF (SEQ IDNO: 71) P 154. NLIGDFEVAEKINAFRAKVH (SEQ ID NO: 72) P 199.GHSVLTAKGMALFGEGKAEF (SEQ ID NO: 73) P 222. FKSSVITLNTNAELFNQSDI (SEQ IDNO: 74) P 240. FPDLGQEVALNANTKNQKIR (SEQ ID NO: 75) or an active site ofone or more of these peptides. In Table 1 above, the following is due:A. Fragments that produce high levels of IgG antibodies to MDA-modifiedpeptides (n = 3), B. Fragments that produce high levels of IgMantibodies, but no difference between native and MDA-modified peptides(n = 9), C. Fragments that produce high levels of IgG antibodies, but nodifference between native and MDA-modified peptides (n = 2), D.Fragments that produce high levels of IgG antibodies to MDA-modifiedpeptides and at least twice as much antibodies in the NHP-pool ascompared to the AHP-pool (n = 5), E. Fragments that produce high levelsof IgM antibodies to MDA-modified peptides and at least twice as muchantibodies in the NHP-pool as compared to the AHP-pool (n = 11), and F.Fragments that produce high levels of IgG antibodies, but no differencebetween intact and MDA-modified peptides but at least twice as muchantibodies in the AHP-pool as compared to the NHP-pool (n = 7).

The present invention relates to the use of at least one recombinanthuman antibody or an antibody fragment thereof directed towards at leastone oxidized fragment of apolipoprotein B in the manufacture of apharmaceutical composition for therapeutical or prophylactical treatmentof atherosclerosis by means of passive immunization.

Further the invention relates to the recombinant preparation of suchantibodies, as well as the invention relates to method for passiveimmunization using such antibodies raised using an oxidizedapolipoprotein B fragment, as antigen, in particular a fragment asidentified above.

The present invention utilises an isolated antibody fragment library togenerate specific human antibody fragments against oxidized, inparticular MDA modified peptides derived from Apo B100. Identifiedantibody fragments with desired characteristics may then rebuilt intofull length human immunoglobulin to be used for therapeutic purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are ELISA results from Screen II;

FIGS. 2A-2F are graphs of dose response for ELISAs;

FIG. 3 are the DNA sequences of various regions;

FIGS. 4A and 4B are light and heavy-chain vectors;

FIG. 5 is a graph of ELISA results;

FIG. 6 is a graph of Oil Red O Stained area in aortas;

FIG. 7 is a graph of Oil Red O stained area in aortas versus antibodyproduct;

FIGS. 8 a and 8 b are graphs of LDL uptake; and

FIG. 9 are graphs of the Ratio MDA/na LDL and ApoB.

DETAILED DESCRIPTION OF THE INVENTION

Below will follow a detailed description of the invention exemplifiedby, but not limited to, human antibodies derived from an isolatedantibody fragment library and directed towards two MDA modified peptidesfrom ApoB 100.

EXAMPLE 1 Selection of scFv Against MDA Modified Peptides IEIGL EGKGFEPTLE ALFGK (SEQ ID NO: 70) (P45, Table 1) and KTTKQ SFDLS VKAQY KKNKH(SEQ ID NO: 52) (P210, Table 1)

The target antigens were chemically modified to carry Malone-di-aldehyde(MDA) groups on lysines and histidines. The modified peptides weredenoted IEI (P45) and KTT (P210).

Selections were performed using BioInvent's n-CoDeR™scFv library forwhich the principle of construction and production have been describedin Soderlind et al. 2000, Nature BioTechnology. 18, 852-856. Briefly,CDRs are isolated from human immunoglobulin genes and are shuffled intoa fixed framework. Thus variability in the resulting immunoglobulinvariable regions is a consequence of recombination of all six CDRs intothe fixed framework. The framework regions are all germline and areidentical in all antibodies. Thus variability is restricted to the CDRswhich are all natural and of human origin. The library containsapproximately 2×10¹⁰ independent clones and a 2000 fold excess of cloneswere used as input for each selection. Selections were performed inthree rounds. In selection round 1, Immunotubes (NUNC maxisorb 444202)were coated with 1.2 ml of 20 μg/ml MDA-modified target peptides in PBS(137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄) with end overend agitation at +4° C. over night. The tubes were then blocked withTPBSB5% (5% BSA, 0.05% Tween 20, 0.02% sodium Azide in PBS) for 30minutes and washed twice with TPBSB3% (3% BSA, 0.05% Tween 20, 0.02%sodium Azíde in PBS) before use. Each target tube was then incubatedwith approximately 2×10¹³ CFU phages from the n-CodeR™ library in 1.8 mlTPBSB3% for 2 h at room temperature, using end over end agitation. Thetubes were then washed with 15×3 ml TPBSB3% and 2×1 ml PBS before thebound phages were eluted with 1 ml/tube of 2 mg/ml trypsin (Roche,109819) for 30 minutes at room temperature. This procedure takesadvantage of a specific trypsin site in the scFv-fusion protein torelease the phage from the target. The reaction was stopped by theaddition of 100 μl of Aprotein (0.2 mg/ml, Roche, cat.236624), and theimmunotubes were washed with 300 ul PBS, giving a final volume of 1.4ml.

For amplification of the selected phage E. Coli HB101F′ cells were grownexponentially in 10 ml of LB medium (Merck, cat. 1.10285) to OD₆₀₀=0.5and infected with the selected and eluted phage principally as described(Soderlind et al., 2000, Nature BioTechnol. 18, 852-856. The resultingphage supernatant was then precipitated by addition of ¼ volume of 20%PEG₆₀₀₀ in 2.5 M NaCl and incubated for 5 h at +4° C. The phages werethen pelleted by centrifugation for 30 minutes, 13000×g, re-suspended in500 μl PBS and used in selection round 2.

The amplified phagestock was used in selection round 2 in a final volumeof 1.5 ml of 5% BSA, 0.05% Tween 20, 0.02% sodium Azfde in PBS. Peptidewithout MDA modification (4×10⁻⁷ M) was also included for competitionagainst binders to MDA-unmodified target peptide. The mixture wasincubated in immunotubes prepared with antigen as described above,except that the tubes were blocked with 1% Casein instead of TPBSB3%.The incubations and washing of the immunotubes were as described forselection 1. Bound phages were then eluted for 30 minutes using 600 μlof 100 mM Tris-Glycine buffer, pH 2.2. The tubes were washed withadditional 200 μl glycin buffer and the eluates were pooled and thenneutralised with 96 μl of 1 M Tris-HCl, pH 8.0. The samples werere-natured for 1 h at room temperature and used for selection round 3.

For selection round 3, BSA, Tween 20 and Sodium Azide were added to therenaturated phage pool to a final concentration of 3%, 0.05% and 0.02%,respectively. Competitor peptides, MDA modified unrelated peptides aswell as native target peptides without modification were added to aconcentration of 1×10⁻⁷M. The phage mixtures (1100 μl) were added toimmunotubes coated with target antigen as described in selection 1 andincubated over night at 4° C. with agitation. The tubes were then washedwith 3×3 ml TPBSB 3%, 5×3 ml PBS and eventually bound phages were elutedusing trypsin as described in selection round 1 above. Each eluate wasinfected to 10 ml of logarithmically growing HB101F′ in LB containing100 μg/ml ampicillin, 15 μg/ml tetracycline, 0.1% glucose, and grownover night at 30° C., 200 rpm in a shaker incubator.

The over night cultures were used for mini scale preparation of plasmidDNA, using Biorad mini prepp Kit (Cat. 732 6100). To remove the phagegene III part from the expression vector, 0.25 μg of the plasmid DNA wascut for 2 h at 37° C. using 2.5 U Eag-1 (New England Biolabs™, cat.R050) in the buffer recommended by the supplier. The samples were thenheat inactivated for 20 minutes at 65° C. and ligated over night at 16°C. using 1 U T4 DNA ligase in 30 μl of 1×ligase buffer (Gibco/BRL). Thisprocedure will join two Eag-1 sites situated on opposite sides of thephage gene III fragment, thus creating a free scFv displaying a terminal6×his tag. After ligation the material was digested for 2 h at 37° C. ina solution containing 30 ul ligation mix, 3.6 μl 10×REACT3 stock, 0.4 μl1 M NaCl, 5 μl H₂O₂, in order to destroy clones in which the phage geneIII segment had been religated. Twenty (20) ng of the final product weretransformed into chemical competent Top10F′ and spread on 500 cm² Q-trayLA-plates (100 μg/ml Amp, 1% glucose), to enable the picking of singlecolonies for further screening.

Screening of the n-CoDeR™scFv Library for Specific Antibody FragmentsBinding t0 MDA Modified Peptides from Anolinonrotein B-100

In order to identify scFv that could discriminate between MDA modifiedIEI (P45) peptide and native IEI and between MDA modified KTT (P210) andnative KTT respectively screenings were performed on bacterialsupernatants from selected scFv expressing clones.

Colony picking of single clones, expression of scFv and screening number1 was performed on BioInvent's automatic system according to standardmethods. 1088 and 831 single clones selected against the MDA modifiedIEI and KTT peptides respectively were picked and cultured and expressedin micro titre plates in 100 μl LB containing 100 μg ampicillin/ml.

For screening number 1 white Assay plates (Greiner 655074) were coatedwith 54 pmol peptide/well in coating buffer (0.1 M Sodium carbonate, pH9.5), either with MDA modified peptide which served as positive targetor with corresponding unmodified peptide which served as non target. Inthe ELISA the expressed scFv were detected through a myc-tag situatedC-terminal to the scFv using 1 μg/ml of anti-c-myc monoclonal (9E10Roche 1667 149) in wash buffer. As a secondary antibody Goat-anti-mousealkaline phosphatase conjugate (Applied Biosystems Cat # AC32ML) wasused at 25000 fold dilution. For luminescence detection CDP-Star Readyto use with Emerald II™ Tropix® (Applied Biosystems Cat # MS100RY) wereused according to suppliers recommendation.

ScFv clones that bound MDA modified peptide but not native peptide werere expressed as described above and to screening another time in aluminescent ELISA (Table 2 and FIG. 1). Tests were run both againstdirectly coated peptides (108 pmol/well coated with PBS) and the morephysiological target, LDL particles (1 μg/well coated in PBS+1 mM EDTA)containing the ApoB-100 protein with and without MDA modification wereused as targets. Positive clones were those that bound oxidised LDL andMDA modified peptide but not native LDL or peptide. The ELISA wasperformed as above except that the anti-His antibody (MaB050 RαD) wasused as the detection antibody. Twelve IEI clones and 2 KTT clones werefound to give more than three fold higher luminescence signal at 700 nmfor the MDA modified form than for the native form both for the peptideand LDL.

The identified clones were further tested through titration against afixed amount (1 μg/well) of MDA LDL and native LDL in order to evaluatethe dose response of the scFv (FIG. 2).

TABLE 2 Screening results. The number of clones tested in each screeningstep for each target. The scored hits in percent are shown withinbrackets. Target IEI KTT Screening Tested Clones 1088 831 number 1Scored Hits 64 33 (%) (5.9%) (4.0%) Screening Tested Clones 64 33 number2 Scored Hits 12 2 (%) (1.1%) (0.2%) Dose Tested Clones 12 2 responseScored Hits 8 2 (%) (0.7%) (0.2%)

The sequences of the chosen scFv clones were determined in order to findunique clones. Bacterial PCR was performed with the Boeringer MannheimExpand kit using primers 5′-CCC AGT CAC GAC GTT GTA AAA CG-3′ (SEQ. ID.NO: 76) and 5′-GAA ACA GCT ATG AAA TAC CTA TTG C-3′ (SEQ. ID. No: 77)and a GeneAmp PCR system 9700 (PE Applied system) using the temperaturecycling program 94° C. 5 min, 30 cycles of 94° C. 30 s, 52° C. for 30 sand 68° C. for 2 min and finally 5 min at 68 min. The sequencingreaction was performed with the bacterial PCR product (five folddiluted) as template, using Big Dye® Terminator mix from PE AppliedBiosystems and the GeneAmp® PCR system 9700 (PE Applied system) and thetemperature cycling program 25 cycles of 96° C. 10 s, 50° C. for 5 s and60° C. for 4 min. The extension products were purified according to thesupplier's instructions and the separation and detection of extensionproducts was done by using a PRISM® 3100 Genetic analyser (PE AppliedBiosystems). The sequences were analysed by the in house computerprogram. From the sequence information homologous clones and clones withinappropriate restriction sites were excluded, leaving six clones forIgG conversion. The DNA sequence of the variable heavy (VH) and variablelight (VL) domains of the finally selected clones are shown in FIG. 3.

EXAMPLE 2 Transfer of Genes Encoding the Variable Parts of Selected scFvto Full Length Human IgG1 Vestors

Bacteria containing scFv clones to be converted to Ig-format were grownover night in LB supplemented with 100 μg/ml ampicillin. Plasmid DNA wasprepared from over night cultures using the Quantum Prep, plasmidminiprep kit from Biorad (# 732-6100). The DNA concentration wasestimated by measuring absorbance at 260 nm, and the DNA was diluted toa concentration of 2 ng/μl.

VH and VL from the different scFv-plasmids were PCR amplified in orderto supply these segments with restriction sites compatible with theexpression vectors (see below). 5′ primers contain a BsmI and 3′ primerscontain a BsiWI restriction enzyme cleavage site (shown in italics). 3′primers also contained a splice donor site (shown in bold).

Primers for amplification of VH-segments:

(SEQ. ID. NO: 13) 5′VH: 5′-GGTGTGCATTCCGAGGTGCAGCTGTTGGAG (SEQ. ID. NO:14) 3′VH: 5′-GACGTACG ACTCACCTGAGCTCACGGTGACCAG

Primers for amplification of VL-segments:

(SEQ. ID. NO: 15) 5′VL: 5′-GGTGTGCATTCCCAGTCTGTGCTGACTCAG (SEQ. ID. NO:16) 3′VL: 5′-GACGTACGTTCTACTCACCTAGGACCGTCAGCTT

PCR was conducted in a total volume of 50 μl, containing long templateDNA, 0.4 μM 5′ primer, 0.4 μM 3′ primer and 0.6 mM dNTP (Roche, #1 969064). The polymerase used was Expand long template PCR system (Roche # 1759 060), 3.5μ per reaction, together with each of the supplied buffersin 3 separate reactions. Each PCR amplification cycle consisted of adenaturing step at 94° C. for 30 seconds, an annealing step at 55° C.for 30 seconds, and an elongating step at 68° C. for 1.5 minutes. Thisamplification cycle was repeated 25 times. Each reaction began with asingle denaturing step at 94° C. for 2 minutes and ended with a singleelongating step at 68° C. for 10 minutes. The existence of PCR productwas checked by agarose gel electrophoresis, and reactions containing thesame amplified material (from reactions with different buffers) werepooled. The PCR amplification products were subsequently purified byspin column chromatography using S400-HR columns (Amersham-PharmaciaBiotech # 27-5240-01).

Four (4) μl of from each pool of PCR products were used for TOPO TAcloning™ (pCR 2.1 TOPO®, InVitrogen #K4550-01) according to themanufacturers recommendations. Bacterial colonies containing plasmidswith inserts were grown over night in LB supplemented with 100 μg/mlampicillin and 20 μg/ml kanamycin. Plasmid DNA was prepared from overnight cultures using the Quantum Prep, plasmid miniprep kit from Biorad(# 732-6100). Plasmid preparations were purified by spin columnchromatography using S400-HR columns (Amersham-Pharmacia Biotech #27-5240-01). Three plasmids from each individual VH and VL cloning weresubjected to sequence analysis using BigDye® Cycle Sequencing (PerkinElmer Applied Biosystem, # 4303150). The cycle sequencing programconsisted of a denaturing step at 96° C. for 10 seconds, an annealingstep at 50° C. for 15 seconds, and an elongating step at 60° C. for 4minutes. This cycle was repeated 25 times. Each reaction began with asingle denaturing step at 94° C. for 1 minute. The reactions wereperformed in a volume of 10 μl consisting of 1 μM primer(5′-CAGGAAACAGCTATGAC) (SEQ ID. NO: 78), 3 μl plasmid DNA and 4 μl BigDye® reaction mix. The reactions were precipitated according to themanufacturer's recommendations, and samples were run on a ABI PRISM®3100 Genetic Analyzer. Sequences were compared to the original scFvsequence using the alignment function of the OMIGA sequence analysissoftware (Oxford Molecular Ltd).

Plasmids containing VH and VL segments without mutations wererestriction enzyme digested. To disrupt the pCR 2.1 TOPO® vector,plasmids were initially digested with DraI (Roche # 1 417 983) at 37° C.for 2 hours. Digestions were heat inactivated at 70° C. for 20 minutesand purified by spin column chromatography using S400-HR columns(Amersham-Pharmacia Biotech # 27-5240-01). The purified DraI digestionswere subsequently digested with BsmI (Roche # 1 292 307) and BsiWI(Roche # 1 388 959) at 55° C. over night. Digestions were purified usingphenol extraction and precipitation. The precipitated DNA was dissolvedin 10 μl H₂O and used for ligation.

The expression vectors were obtained from Lars Norderhaug (J. Immunol.Meth. 204 (1997) 77-87). After some modifications, the vectors (FIG. 4)contain a CMV promoter, an Ig-leader peptide, a cloning linkercontaining BsmI and BsiWI restriction sites for cloning of VH/VL,genomic constant regions of IgG1(heavy chain (HC) vector) or lambda(light chain (LC) vector), neomycin (HC vector) or methotrexate (LCvector) resistance genes for selection in eukaryotic cells, SV40 andColEI origins of replication and ampicillin (HC vector) or kanamycin (LCvector) resistance genes for selection in bacteria.

The HC and LC vectors were digested with BsmI and BsiWI, phosphatasetreated and purified using phenol extraction and precipitation. Ligationwere set up at 16° C. over night in a volume of 10 μl, containing 100 ngdigested vector, 2 μl digested VH/VL-pCR 2.1 TOPO® vector (see above), 1U T4 DNA ligase (Life Technologies, # 15224-025) and the suppliedbuffer. 2 μl of the ligation mixture were subsequently transformed into50 μl chemocompetent top10F′ bacteria, and plated on selective (100μg/ml ampicillin or 20 μg/ml kanamycin) agar plates.

Colonies containing HC/LC plasmids with VH/VL inserts were identified bycolony PCR:

(SEQ ID NO: 17) Forward primer: 5′-ATGGGTGACAATGACATC (SEQ ID NO: 18)Reverse primer: 5′-AAGCTTGCTAGCGTACG

PCR was conducted in a total volume of 20 μl, containing bacterias, 0.5μM forward primer, 0.5 μM reverse primer and 0.5 mM dNTP (Roche, #1 969064). The polymerase used was Expand long template PCR system (Roche # 1759 060), 0.7 Upper reaction, together with the supplied buffer #3. EachPCR amplification cycle consisted of a denaturing step at 94° C. for 30seconds, an annealing step at 52° C. for 30 seconds, and an elongatingstep at 68° C. for 1.5 minutes. This amplification cycle was repeated 30times. Each reaction began with a single denaturing step at 94° C. for 2minutes and ended with a single elongating step at 68° C. for 5 minutes.The existence of PCR product was checked by agarose gel electrophoresis.Colonies containing HC/LC plasmids with VH/VL inserts were grown overnight in LB supplemented with 100 μg/ml ampicillin or 20 μg/mlkanamycin. Plasmid DNA was prepared from over night cultures using theQuantum Prep, plasmid miniprep kit from Biorad (# 732-6100). Plasmidpreparations were purified by spin column chromatography using S400-HRcolumns (Amersham-Pharmacia Biotech # 27-5240-01). To confirm theintegrity of the DNA sequence, three plasmids from each individual VHand VL were subjected to sequence analysis using BigDye® CycleSequencing (Perkin Elmer Applied Biosystem, # 4303150). The cyclesequencing program consisted of a denaturing step at 96° C. for 10seconds, an annealing step at 50° C. for 15 seconds, and an elongatingstep at 60° C. for 4 minutes. This cycle was repeated 25 times. Eachreaction began with a single denaturing step at 94° C. for 1 minute. Thereactions were performed in a volume of 10 μl consisting of 1 μM primer5′-AGACCCAAGCTAGCTTGGTAC (SEQ. ID. No: 79), 3 μl plasmid DNA and 4 μlBig Dye® reaction mix. The reactions were precipitated according to themanufacturer's recommendations, and samples were run on a ABI PRISM®3100 Genetic Analyzer. Sequences were analysed using the OMIGA sequenceanalysis software (Oxford Molecular Ltd). The plasmid DNA was used fortransient transfection of COS-7 cells (see below) and were digested forproduction of a joined vector, containing heavy- and light chain geneson the same plasmid.

Heavy and light chain vectors containing VH and VL segments originatingfrom the same scFv were cleaved by restriction enzymes and ligated: HC-and LC-vectors were initially digested with MunI (Roche # 1 441 337)after which digestions were heat inactivated at 70° C. for 20 minutesand purified by spin column chromatography using S200-HR columns(Amersham-Pharmacia Biotech # 27-5120-01). HC-vector digestions weresubsequently digested with NruI (Roche # 776 769) and LC-vectordigestions with Bst1107I (Roche # 1 378 953). Digestions were then heatinactivated at 70° C. for 20 minutes and purified by spin columnchromatography using S400-HR columns (Amersham-Pharmacia Biotech #27-5240-01). 5 μl of each digested plasmid were ligated at 16° C. overnight in a total volume of 20 μl, containing 2 U T4 DNA ligase (LifeTechnologies, # 15224-025) and the supplied buffer. 2 μl of the ligationmixture were subsequently transformed into 50 μl chemocompetent top10F′bacteria, and plated on selective (100 μg/ml ampicillin and 20 μg/mlkanamycin) agar plates.

Bacterial colonies were grown over night in LB supplemented with 100μg/ml ampicillin and 20 μg/ml kanamycin. Plasmid DNA was prepared fromover night cultures using the Quantum Prep, plasmid miniprep kit fromBiorad (# 732-6100). Correctly joined vectors were identified byrestriction enzyme digestion followed by analyses of fragment sizes byagarose gel-electrophoreses.

Plasmid preparations were purified by spin column chromatography usingS400-HR columns (Amersham-Pharmacia Biotech # 27-5240-01) and used fortransient transfection of COS-7 cells.

COS-7 cells (ATCC # CRL-1651) were cultured at 37° C. with 5% CO₂ inDulbeccos MEM, high glucose+GlutamaxI™ (Invitrogen # 31966021),supplemented with 0.1 mM non-essential amino acids (Invitrogen #11140035) and 10% fetal bovine sera (Invitrogen # 12476-024, batch #1128016). The day before transfection, the cells were plated in 12-wellplates (Nunc™, # 150628) at a density of 1.5×10⁵ cells per well.

Prior to transfection, the plasmid DNA was heated at 70° C. for 15minutes. Cells were transfected with 1 μg HC-plasmid+1 μg LC-plasmid, or2 μg joined plasmid per well, using Lipofectamine™ 2000 Reagent(Invitrogen, # 11668019) according to the manufacturers recommendations.24 hours post transfection, cell culture media was changed and the cellswere allowed to grow for 5 days. After that, medium was collected andprotein production was assayed for using ELISA.

Ninetysix (96)-well plates (Costar # 9018, flat bottom, high binding)were coated at 4° C. over night by adding 100 μl/well rabbit anti-humanlamda light chain antibody (DAKO, # A0193) diluted 4000 times in coatingbuffer (0.1M sodium carbonate, pH 9.5). Plates were washed 4 times inPBS containing 0.05% Tween 20 and thereafter blocked with 100 μl/wellPBS+3% BSA (Albumin, fraction V, Roche # 735108) for 1 h. at roomtemperature. After washing as above, 100 μl/well of sample were addedand incubated in room temperature for 1 hour. As a standard forestimation of concentration, human purified IgG1 (Sigma, # 15029) wasused. Samples and standard were diluted in sample buffer (1×PBScontaining 2% BSA and 0.5% rabbit serum (Sigma # R4505). Subsequently,plates were washed as described above and 100 μl/well of rabbitanti-human IgG (γ-chain) HRP-conjugated antibody (DAKO, # P214) diluted8000 times in sample buffer was added and incubated at room temperaturefor 1 hour. After washing 8 times with PBS containing 0.05% Tween 20,100 μl/well of a substrate solution containing one OPD tablet (10 mg,Sigma # P8287,) dissolved in 15 ml citric acid buffer and 4.5 μl H₂O₂(30%) was added. After 10 minutes, the reaction was terminated by adding150 μl/well of 1M HCl. Absorbance was measured at 490-650 nm and datawas analyzed using the Softmax software.

Bacteria containing correctly joined HC- and LC-vectors were grown overnight in 500 ml LB supplemented with ampicillin and kanamycin. PlasmidDNA was prepared from over night cultures using the Quantum Prep,plasmid maxiprep kit from Biorad (# 732-6130). Vectors were linearizedusing PvuI restriction enzyme (Roche # 650 129). Prior to transfection,the linearized DNA was purified by spin column chromatography usingS400-HR columns (Amersham-Pharmacia Biotech # 27-5240-01) and heated at70° C. for 15 minutes.

EXAMPLE 3 Stable Transfection of NS0 Cells Expressing Antibodies AgainstMDA Modified Peptides Form Apolipoprotein B-100

NS0 cells (ECACC no. 85110503) were cultured in DMEM (cat nr 31966-021,Invitrogen) supplemented with 10% Fetal Bovine Serum (cat no. 12476-024,lot: 1128016, Invitrogen) and 1×NEAA (non-essential amino acids, cat no.11140-053, Invitrogen). Cell cultures are maintained at 37° C. with 5%CO₂ in humidified environment.

DNA constructs to be transfected were four constructs of IEI specificantibodies (IEI-A8, IEI-D8, IEI-E3, IEI-G8), two of KTT specificantibodies (KTT-B8, KTT-D6) and one control antibody (JFPA12). The daybefore transfection, the cells were trypsinized and counted, beforeplating them in a T-75 flask at 12×10⁶ cells/flask. On the day oftransfection, when the cells were 85-90% confluent, the cells wereplated in 15 ml DMEM+1×NEAA+10% FBS (as above). For each flask of cellsto be transfected, 35-40 μg of DNA were diluted into 1.9 ml of OPTI-MEM®I Reduced Serum Medium (Cat no. 51985-026, lot: 3062314, Invitrogen)without serum. For each flask of cells, 114 μl of Lipofectamine™ 2000Reagent (Cat nr. 11668-019, lot: 1116546, Invitrogen) were diluted into1.9 ml OPTI-MEM® I Reduced Serum Medium in another tube and incubatedfor 5 min at room temperature. The diluted DNA was combined with thediluted Lipofectamine™ 2000 Reagent (within 30 min) and incubated atroom temperature for 20 min to allow DNA-LF2000 Reagent complexes toform.

The cells were washed with medium once and 11 ml DMEM+1×NEAA+10% FBSwere added. The DNA-LF2000 Reagent complexes (3.8 ml) were then addeddirectly to each flask and gently mixed by rocking the flask back andforth. The cells were incubated at 37° C. in a 5% CO₂ incubator for 24h.

The cells were then trypsinized and counted, and subsequently plated in96-well plates at 2×10⁴ cells/well using five 96-well plates/construct.Cells were plated in 100 μl/well of DMEM+1×NEAA+10% FBS (as above)containing G418-sulphate (cat nr.10131-027, lot: 3066651, Invitrogen) at600 μg/ml. The selection pressure was kept unchanged until harvest ofthe cells.

The cells were grown for 12 days and assayed for antibody productionusing ELISA. From each construct cells from the 24 wells containing thehighest amounts of IgG were transferred to 24-well plates and wereallowed to reach confluency. The antibody production from cells in thesewells was then assayed with ELISA and 5-21 pools/construct were selectedfor re-screening (Table 3). Finally cells from the best 1-4 wells foreach construct were chosen. These cells were expanded successively incell culture flasks and finally transferred into triple layer flasks(500 cm2) in 200 ml of (DMEM+1xNEAA+10% Ultra low IgG FBS (cat.no.16250-078, lot.no. 113466, Invitrogen)+G418 (600 μg/ml)) for antibodyproduction. The cells were incubated for 7-10 days and the supernatantswere assayed by ELISA, harvested and sterile filtered for purification.

EXAMPLE 4 Production and Purification of Human IgG1

Supernatants from NS0 cells transfected with the different IgG1antibodies were sterile filtered using a 0.22 μm filter and purifiedusing an affinity medium MabSelect™ with recombinant protein A, (Cat.No. 17519901 Amersham Biosciences).

Bound human IgG1 was eluted with HCL-glycine buffer pH 2.8. The eluatewas collected in 0.5 ml fractions and OD₂₈₀ was used to determinepresence of protein. The peak fractions were pooled and absorbance wasmeasured at 280 nm and 320 nm. Buffer was changed through dialysisagainst a large volume of PBS. The presence of endotoxins in thepurified IgG-1 preparations was tested using a LAL test (QCL-1000®, cat.No. 50-647U Bio Whittaker). The samples contained between 1 and 12 EU/mlendotoxin. The purity of the preparations were estimated to exceed 98%by PAGE analysis.

TABLE 3 Summary of Production and Purification of human IgG1 Volumeculture Total IgG1 in Total IgG1 Clone supernatant supernatant Purifiedname (ml) (mg) (mg) Yield (%) IEI-A8 600 68 42 61.8 IEI-D8 700 45 2146.7 IEI-E3 700 44.9 25.6 60 IEI-G8 600 74 42.4 57.3 KTT-B8 1790 77.337.6 48.6 KTT-D6 1845 47.8 31.8 66.5 JFPA12 2000 32.2 19.2 59.6

The purified IgG1 preparations were tested in ELISA for reactivity toMDA modified and un-modified peptides (FIG. 5) and were then used infunctional in vitro and in vivo studies.

EXAMPLE 5 Analysis of Possible Anti-Atherogenic Effect of Antibodies arePerformed Both in Experimental Animals and in Cell Culture Studies

-   -   1. Effect of antibodies on atherosclerosis in apolipoprotein E        knockout (apo E−) mice. Five weeks old apo E−mice are fed a        cholesterol-rich diet for 15 weeks. This treatment is known to        produce a significant amount of atherosclerotic plaques in the        aorta and carotid arteries. The mice are then given an        intraperitoneal injection containing 500 μg of the respective        antibody identified above. Control mice are given 500 μg of an        irrelevant control antibody or PBS alone. Treatments are        repeated after 1 and 2 weeks. The mice are sacrificed 4 weeks        after the initial antibody injection. The severity of        atherosclerosis in the aorta is determined by Oil Red O staining        of flat preparations and by determining the size of subvalvular        atherosclerotic plaques. Collagen, macrophage and T cell content        of subvalvular atherosclerotic plaques is determined by Masson        trichrome staining and cell-specific immunohistochemistry.        Quantification of Oil Red O staining, the size of the        subvalvular plaques, trichrome staining and immunohistochemical        staining is done using computer-based image analysis.    -   In a first experiment the effect of the antibodies on        development of atherosclerosis was analysed in apo E−/− mice fed        a high-cholesterol diet. The mice were given three        intraperitoneal injections of 0.5 mg antibody with week        intervals starting at 21 weeks of age, using PBS as control.        They were sacrificed two weeks after the last antibody        injection, and the extent of atherosclerosis was assessed by Oil        Red O staining of descending aorta flat preparations. A        pronounced effect was observed in mice treated with the IEI-E3        antibody, with more than 50% reduction of atherosclerosis as        compared to the PBS group (P=0.02) and to a control group        receiving a human IgG1 antibody (FITC8) directed against a        non-relevant fluorescein isothiocynate (FITC) antigen (P=0.03)        (FIG. 6). The mice tolerated the human antibodies well and no        effects on the general health status of the mice were evident.    -   To verify the inhibitory effect of the IEI-E3 antibody on        development of atherosclerosis we then performed a dose-response        study. The schedule was identical to that of the initial study.        In mice treated with IEI-E3 antibodies atherosclerosis was        reduced by 2% in the 0.25 mg group (n.s.), by 25% in the 0.5 mg        group (n.s.) and by 41% (P=0.02) in the 2.0 mg group as compared        to the corresponding FITC antibody-treated groups (FIG. 7).    -   2. Effect of antibodies on neo-intima formation following        mechanical injury of carotid arteries in apo E− mice. Mechanical        injury of arteries results in development of fibro-muscular        neo-intimal plaque within 3 weeks. This plaque resembles        morphologically a fibro-muscular atherosclerotic plaque and has        been used as one model for studies of the development of raised        lesion. Placing a plastic collar around the carotid artery        causes the mechanical injury. Five weeks old apo E− mice are fed        a cholesterol-rich diet for 14 weeks. The mice are then given an        intraperitoneal injection containing 500 μg of the respective        antibody. Control mice are given 500 μg of an irrelevant control        antibody or PBS alone. The treatment is repeated after 7 days        and the surgical placement of the plastic collar is performed 1        day later. A last injection of antibodies or PBS is given 6 days        after surgery and the animals are sacrificed 15 days later. The        injured carotid artery is fixed, embedded in paraffin and        sectioned. The size of the neo-intimal plaque is measured using        computer-based image analysis.    -   3. Effect of antibodies on uptake of oxidized LDL in cultured        human macrophages. Uptake of oxidized LDL in arterial        macrophages leading to formation of cholesterol-loaded        macrophage foam cells is one of the most characteristic features        of the atherosclerotic plaque. Several lines of evidence suggest        that inhibiting uptake of oxidized LDL in arterial macrophages        represent a possible target for treatment of atherosclerosis. To        study the effect of antibodies on macrophage uptake of oxidized        c are pre-incubated with ¹²⁵I-labeled human oxidized LDL for 2        hours. Human macrophages are isolated from blood donor buffy        coats by centrifugation in Ficoll hypaque followed by culture in        presence of 10% serum for 6 days. The cells are then incubated        with medium containing antibody/oxidized LDL complexes for 6        hours, washed and cell-associated radioactivity determined in a        gamma-counter. Addition of IEI-E3 antibodies resulted in a        five-fold increase in the binding (P=0.001) and uptake (P=0.004)        of oxidized LDL compared to FITC-8 into macrophages, but had no        effect on binding or uptake of native LDL (FIGS. 8 a and 8 b).    -   4. Effect of antibodies on oxidized LDL-dependent cytotoxicity.        Oxidized LDL is highly cytotoxic. It is believed that much of        the inflammatory activity in atherosclerotic plaques is        explained by cell injury caused by oxidized LDL. Inhibition of        oxidized LDL cytotoxicity thus represents another possible        target for treatment of atherosclerosis. To study the effect of        antibodies on oxidized LDL cytotoxicity cultured human arterial        smooth muscle cells are exposed to 100 ng/ml of human oxidized        LDL in the presence of increasing concentrations of antibodies        (0-200 ng/ml) for 48 hours. The rate of cell injury is        determined by measuring the release of the enzyme LDH.

The experiment shown discloses an effect for a particular antibodyraised against a particular peptide, but it is evident to the oneskilled in the art that all other antibodies raised against the peptidesdisclosed will behave in the same manner.

The antibodies of the present invention are used in pharmaceuticalcompositions for passive immunization, whereby the pharmaceuticalcompositions primarily are intended for injection, comprising asolution, suspension, or emulsion of a single antibody or a mixture ofantibodies of the invention in a dosage to provide a therapeutically orprophylactically active level in the body treated. The compositions maybe provided with commonly used adjuvants to enhance absorption of theantibody or mixture of antibodies. Other routes of administration may bethe nasal route by inhaling the antibody/antibody mixture in combinationwith inhalable excipients.

Such pharmaceutical compositions may contain the active antibody in anamount of 0.5 to 99.5% by weight, or 5 to 90% by weight, or 10 to 90% byweight, or 25 to 80% by weight, or 40 to 90% by weight.

The daily dosage of the antibody, or a booster dosage shall provide fora therapeutically or prophylactically active level in the body treatedto reduce or prevent signs and sympthoms of atherosclerosis by way ofpassive immunization. A dosage of antibody according to the inventionmay be 1 μg to 1 mg per kg bodyweight, or more.

The antibody composition can be supplemented with other drugs fortreating or preventing atherosclerosis or heart-vascular diseases, suchas blood pressure lowering drugs, such as beta-receptor blockers,calcium antagonists, diurethics, and other antihypertensive agents.

FIG. 9 shows binding of isolated scFv to MDA modified ApoB100 derivedpeptides and to a MDA modified control peptide of irrelevant sequence.Also depicted are the ratios between binding of the scFv to MDA modifiedand native ApoB100 protein and human LDL respectively. Columns appear inthe order they are defined from top to bottom in right hand column ofthe respective subfigure.

1. A purified or recombinant antibody or antibody fragment capable ofbinding to fragments of apolipoprotein B 100, wherein the antibodycomprises the variable heavy region (V_(H)) encoded by the nucleic acidsequence of SEQ ID NO:27 and the variable light region (V_(L)) encodedby the nucleic acid sequence of SEQ ID NO:28.
 2. The antibody accordingto claim 1 wherein the antibody is capable of binding to an oxidizedfragment of the polypeptide encoded by the nucleic acid sequence of SEQID NO:70.
 3. A pharmaceutical composition comprising an antibody asdefined in claim 1 or 2 for treatment of atherosclerosis by passiveimmunization, which antibody is present in combination with apharmaceutical excipient.